Activation of a cysteine protease in MCF-7 and T47D breast cancer cells during β-lapachone-mediated apoptosis

John J. Pink, Shelly Wuerzberger-Davis, Colleen Tagliarino, Sarah M. Planchon, XiaoHe Yang, Christopher J. Froelich, David A. Boothman

Research output: Contribution to journalArticle

114 Scopus citations

Abstract

β-Lapachone (β-lap) effectively killed MCF-7 and T47D cell lines via apoptosis in a cell-cycle-independent manner. However, the mechanism by which this compound activated downstream proteolytic execution processes were studied. At low concentrations, β-lap activated the caspase-mediated pathway, similar to the topoisomerase I poison, topotecan; apoptotic reactions caused by both agents at these doses were inhibited by zVAD-fmk. However at higher doses of β-lap, a novel non-caspase-mediated 'atypical' cleavage of PARP (i.e., an ~60-kDa cleavage fragment) was observed. Atypical PARP cleavage directly correlated with apoptosis in MCF-7 cells and was inhibited by the global cysteine protease inhibitors iodoacetamide and N- ethylmaleimide. This cleavage was insensitive to inhibitors of caspases, granzyme B, cathepsins B and L, trypsin, and chymotrypsin-like proteases. The protease responsible appears to be calcium-dependent and the concomitant cleavage of PARP and p53 was consistent with a β-lap-mediated activation of calpain. β-Lap exposure also stimulated the cleavage of lamin B, a putative caspase 6 substrate. Reexpression of procaspase-3 into caspase-3-null MCF-7 cells did not affect this atypical PARP proteolytic pathway. These findings demonstrate that β-lap kills cells through the cell-cycle-independent activation of a noncaspase proteolytic pathway. (C) 2000 Academic Press.

Original languageEnglish (US)
Pages (from-to)144-155
Number of pages12
JournalExperimental Cell Research
Volume255
Issue number2
DOIs
StatePublished - Mar 15 2000

Keywords

  • Apoptosis
  • Breast cancer
  • Calpain
  • Caspase
  • PARP
  • Poly(ADP)-ribose polymerase
  • Topotecan
  • β-lapachone

ASJC Scopus subject areas

  • Cell Biology

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    Pink, J. J., Wuerzberger-Davis, S., Tagliarino, C., Planchon, S. M., Yang, X., Froelich, C. J., & Boothman, D. A. (2000). Activation of a cysteine protease in MCF-7 and T47D breast cancer cells during β-lapachone-mediated apoptosis. Experimental Cell Research, 255(2), 144-155. https://doi.org/10.1006/excr.1999.4790